1. Field of Invention
The present invention relates to an apparatus and method for growing anaerobic microorganisms. The apparatus is comprised of a specially designed culture dish which can be reconfigured such as by inverting the dish to produce an anaerobic environment. An oxygen reducing agent such as a biocatalytic oxygen reducing agent can also be incorporated into the media present in the apparatus together, in some circumstances, with a substrate. The biocatalytic oxygen reducing agent and the substrate present in the media react with oxygen enclosed in the culture dish to create an environment suitable for growing and maintaining anaerobic microorganisms.
2. Description of Prior Art
Microorganisms are important to our well being. This is evident in health care, agriculture and industry. To be able to simply and quickly isolate and grow microbes is economically important. For example, being able to quickly and specifically isolate and identify a microbe responsible for infection is important in the human health care field. This basic technique is also important in the agriculture industry. Large scale processing of food requires constant microbial monitoring. The speed and efficiency at which this can be done determines the length of time finished food products must be held in storage before they can be distributed for sale.
Control of the environment is necessary for control of microbial growth. In particular, control of oxygen content in the immediate environment is crucial for microbial growth. Microorganisms can be divided into groups based on their need for, and tolerance of, oxygen. There are those that require oxygen to grow. These are xe2x80x9caerobesxe2x80x9d. Some microorganisms are able to grow with or without oxygen. These are xe2x80x9cfacultative anaerobesxe2x80x9d. Another group of microorganisms can grow only in the presence of very low levels of oxygen. These are the xe2x80x9cmicroaerophilesxe2x80x9d. Finally, some microorganisms can not tolerate oxygen. They are inhibited by it or may be killed by it. These are the xe2x80x9canaerobesxe2x80x9d.
This fundamental property of microorganisms, that is their ability to grow in or tolerate oxygen, is used daily to isolate, grow, and manipulate them. One basic technique in microbiology, is the plating method. This generally involves the use of a dish, developed by Petri (i.e. xe2x80x9cPetri dishxe2x80x9d) in 1880""s, and solidified (agar or gelatin-based) medium.
A Petri dish is usually a round, shallow, flat-bottomed, glass or plastic dish (often e.g. 10 cm diameter) with a vertical side, that cooperates with a similar, slightly larger structure which forms a loosely-fitting lid. Petri dishes are used in microbiology, e.g., for the preparation of plates.
The purpose of the Petri dish is to provide a controlled environment for selectively growing microbes. The dish is sterilized and designed to maintain a sterile environment inside while freely exchanging gases, normally air, with the outside environment.
The medium utilized in conjunction with the Petri dish can be formulated to provide a necessary and selective environment for a specific microorganism. Solid medium in a Petri dish can be prepared using aseptic technique by pouring sterile molten or liquid (agar- or gelatin-based) medium into a Petri dish to a depth of 3-5 mm and allowing it to set. Generally, freshly poured plates to be used for separation and/or generation of microbes should be left for 30 minutes in a 45xc2x0 C. hot-air incubator with the lid partly off so that the surface moisture can evaporate. Such xe2x80x9cdryingxe2x80x9d before inoculation prevents unwanted spreading of the inoculum in the surface film of the moisture.
The solid medium surface inside the dish provides a place to grow microorganisms. By inoculating (or xe2x80x9cplatingxe2x80x9d) the surface of the agar in a controlled way (i.e. xe2x80x9cstreakingxe2x80x9d), single colonies of a microorganism can be obtained. With this technique the microbiologist can separate microbes one from another. Isolation and purification is mandatory to further characterization and study. Using this dish design, a microbiologist can isolate and grow the great majority of microorganisms known today.
Working with microbes that are microaerophiles or anaerobes poses a problem. The culture dishes for these microbes must be incubated in a controlled gaseous environment that lacks oxygen, or at least most of the oxygen, found in air. This is done by placing the culture or Petri dish containing medium inside a container that is sealed from the outside atmosphere. For one or a few dishes, a sealable bag or jar (i.e., xe2x80x9cBrewer Jarxe2x80x9d) is used (Becton Dickinson Microbiology Systems, 1994 Catalog, p 89-p 94). In this case, chemicals and a catalyst (see U.S. Pat. No. 4,287,306 issued Sep. 1, 1982 to Brewer entitled xe2x80x9cApparatus for Generation of Anaerobic Atmospherexe2x80x9d) are placed inside the container that, when activated chemically, reacts with the oxygen in the container, thus removing it. The catalyst is necessary to bring about the reaction at low temperatures in a short time.
In addition, for many culture dishes, a sealed table-top chamber can be used (Anaerobe Systems, San Jose, Calif.). This chamber is evacuated and flushed with inert gases, such as nitrogen and/or carbon dioxide. Sometimes chemicals and a catalyst are used to consume the oxygen inside the chamber and fresh, inert gas is supplied as needed. The microbiologist works with the culture dishes inside of this chamber through ports fitted with gloves. A means is provided for introducing materials into and taking items out of the chamber without breaching the anaerobic environment inside.
Work with microaerophiles and anaerobes under these conditions is labor intensive, difficult, expensive, and time consuming. The microbiologist is often frustrated by having to wait for the slowest growing microbe in order to retrieve all culture dishes from a bag or jar since once the bag or jar is opened, the microbes are exposed to oxygen. A failure in the system can be catastrophic for all of the microbial isolates inside.
To overcome many of these problems (see U.S. Pat. No. 2,348,448 issued May 9, 1944 to Brewer entitled xe2x80x9cApparatus for the Cultivation of Anaerobic and Microaerophilic Organismsxe2x80x9d) Brewer developed a culture dish lid (i.e., xe2x80x9cBrewer Lidxe2x80x9d) that formed a seal between a ring inside the lid with the agar or gelatin-based surface. Within the dish, a very small, defined headspace is formed by the lid and the agar surface. An anaerobic environment is created inside this trapped headspace by reacting oxygen with chemical reducing agents, such as thioglycollate, incorporated in the medium. The limited volume of the headspace is important to the function of the Brewer Lid.
However, a number of drawbacks exist in the use of the Brewer Lid. The capacity and the rate for oxygen removal is limited by the sensitivity of the microorganism to the chemical reducing agent in the medium (see xe2x80x9cMechanism of Growth Inhibitory Effect of cysteine on Escherichia coli.xe2x80x9d of Kari, et al., J. Gen. Microbiol., 68, 1971, p. 349 and xe2x80x9cMethods for General and Molecular Bacteriologyxe2x80x9d, Editor: Gerherdt, American Society for Microbiology, 1994, p. 146.). Moreover, the lid is made of heavy glass and is expensive. It is available today (Kimble Glass Company, Vineland, N.J.), but is not widely used because of serious limitations that include cost, handling difficulties, and poor response of anaerobic microorganisms.
Another limitation is caused by the material of construction. The glass Brewer Lid is made very heavy to insure a good seal between the ring inside the Brewer Lid and the agar surface. Cultures dish bottoms fitted with the heavy Brewer Lid are not easy to handle or to move about. They can not be stacked inside an incubator. Thus, precious incubator space is wasted. Stacked dishes crush the agar medium of the lowest dishes in the stack, because of the weight of the dishes above them. This causes the headspace above the agar to collapse resulting in contact between the inside of the Brewer Lid and the agar surface. When this happens, the microbial growth on the surface is spread out and separation of individual colonies is lost. Motile microbes will migrate and further frustrate separation.
Because of their weight and material of construction, Brewer Lids do not lend themselves to commercial production of pre-made agar or gelatin-based plates. The commercial process requires assembly line filling of the dishes, packaging the filled dishes in stacks, and handling and storing these dishes. Pre-made agar plates are widely used in clinical microbiological laboratories. This limitation of the Brewer Lid is economically significant.
The headspace inside the Brewer Lid formed by the lid and agar surface is very small. This limited headspace is determined by the ability of the chemical reducing agent (H2S, cysteine, thioglycollate, etc.) to reduce oxygen in the headspace. The amount of chemical reducing agent used in the medium in turn is constrained by anaerobic microorganism""s sensitivity to it. The sum of these limitations is a very small head space that imparts severe problems to the function of the Brewer Lid for its intended purpose, i.e. to grow anaerobic and microaerophilic microorganisms.
Another limitation of the Brewer Lid is that the very limited head space can not hold much moisture. Fresh agar medium is generally greater than 98 percent water. Upon incubation, water in the medium evaporates and condenses upon the upper surface of the inside of the lid. This condensate can become sufficient to fall to the agar surface and to flood it. Under such conditions, the plate is ruined and can not be used for isolation and purification of the microbe.
The very limited headspace imposes still more limitations on the Brewer Lid. No provision is made to incorporate CO2 into the headspace above the agar surface. This is important for the rapid growth of some microorganisms and may be required by others. Yet this feature should be made optional for the microbiologist, because for some uses of the culture dish the microbiologist may not want to include CO2 in the headspace. Reports show that CO2 can change the pH of the medium it contacts. This in turn can interfere with the determination of susceptibility to some antibiotics (see xe2x80x9cEffect of CO2 on Susceptibilities of Anaerobes to Erythromycin, Azithromycin, Clarithromycin, and Roxithromycinxe2x80x9d, Spangler, et al., Antimicrob. Agents Chemotherapy, 38, p. 20, 1994). Since CO2 is generated in anaerobic jars and bags by commercial catalysts products, this problem is commonly encountered. CO2 is a component of the gas used to flush anaerobic chambers and incubators too.
Another desired feature for a self contained culture dish is an indicator to show that the headspace is anaerobic. These features are difficult to impossible to include in the Brewer Lid because of the very small space between inside the lid top and the agar surface.
Several attempts have been made to design a culture dish that provides a self-contained environment for growing anaerobic microorganisms (see U.S. Pat. No. 2,701,229 issued Feb. 1, 1955 to Scherr entitled xe2x80x9cApparatus for the Cultivation of Microorganismsxe2x80x9d; U.S. Pat. No. 3,165,450 issued Jan. 12, 1965 to Scheidt entitled xe2x80x9cAnaerobic Culturing Devicexe2x80x9d; U.S. Pat. No. 4,294,924 issued Oct. 13, 1981 to Pepicelli, et al. entitled xe2x80x9cMethod and Container for Growth of Anaerobic Microorganismsxe2x80x9d; U.S. Pat. No. 4,299,921 issued Nov. 10, 1981 to Youssef entitled xe2x80x9cProlonged Incubation Microbiological Apparatus and Filter Gaskets Thereofxe2x80x9d; and U.S. Pat. No. 4,859,586 issued Aug. 8, 1989 to Eisenberg entitled xe2x80x9cDevice for Cultivating Bacteriaxe2x80x9d). The fact that the Brewer Lid and none of these inventions are commonly or commercially available or used widely by microbiologists today, attest to their limitations and shortcomings. The need to simplify and reduce the cost for isolating and growing anaerobic and microaerophilic microorganisms still exists today.
It is therefore an object of the present invention to provide an improved apparatus and method for cultivating and/or enumerating anaerobic microorganisms which obviate the above-mentioned disadvantages of the prior art.
Another object of the present invention is to provide an improved anaerobic culturing apparatus which is extremely simple, inexpensive and easy to use and wherein the proper anaerobic environment is produced and maintained in an extremely efficient manner.
These and other additional objects and advantages of the present invention will become apparent from the following description of the invention.
The present inventors have designed a novel culture apparatus or dish in order to eliminate many of the difficulties observed in the prior art. It has been found that the use of the new culture dish (i.e., xe2x80x9cOxyDish(trademark)xe2x80x9d) together with an oxygen reducing agent (preferably a biocatalytic oxygen reducing agent) and, in some instances, a substrate, produces a controlled, self-contained environment for isolating, enumerating, identifying and growing facultative aerobes, microaerophiles and anaerobes. The use of the specially designed culture dish along with an oxygen reducing agent makes possible the design and function of a culture dish that utilizes some features of the Brewer Lid, but overcomes its limitations and makes possible novel and improved characteristics.
In this regard, the present invention is directed to a specifically designed culture dish with a dish top or cover that contains a sealing ring on the inside upon which the solid media surface in the bottom dish rests when the dish is inverted to form a media-ring seal. The seal so formed traps the gas in the headspace between the media surface and the inside of the dish top or cover. In addition, an oxygen reducing agent, such as a biocatalytic oxygen reducing agent, can be incorporated into the media present in the culture dish together, in some instances, with a substrate which reacts with oxygen in the media and the headspace to create an environment suitable for growing anaerobic microorganisms.
The preferred biocatalytic oxygen reducing agent (see xe2x80x9cA Novel Approach to the Growth of Anaerobic Microorganismsxe2x80x9d of Adler, et al., Biotechnol. Bioegn. Symp. 11, J. Wiley and Sons, New York, 1981, p. 533 and U.S. Pat. No. 4,476,224 issued Oct. 9, 1984 to Adler entitled xe2x80x9cMaterial and Method for Promoting the Growth of Anaerobic Bacteriaxe2x80x9d) utilized in the invention is comprised of oxygen scavenging membrane fragments which contain an electron transport system which reduces oxygen to water in the presence of a hydrogen donor. These oxygen scavenging membrane fragments can be derived from the cytoplasmic membranes of bacteria (U.S. Pat. No. 4,476,224) and/or from the membranes of mitochondrial organelles of a large number of higher non-bacterial organisms. Other known biocatalytic oxygen reducing agents such as glucose oxidase, alcohol oxidase, etc. can also be utilized.
The biocatalytic oxygen reducing agents suitable for use in the invention are non-toxic to microorganisms. Being catalysts, they are dynamic and highly efficient at reducing the oxygen in the trapped headspace in the specially designed culture dish. The biocatalytic oxygen reducing agents use substrates that are commonly found in microbiological media and that are natural to microorganisms to effect this reaction. The products produced from this reaction are also natural and non-toxic to microorganisms. The use of the biocatalytic oxygen reducing agents makes possible the opening and closing of this dish several times and the agents continue to reduce the oxygen trapped in the headspace after each occurrence.
The culture dish (xe2x80x9cOxyDish(trademark)xe2x80x9d) containing the oxygen reducing agent provides a means to work with microorganisms free of the complications and expense of anaerobic bags, jars, incubators, or chambers. Each dish is light in weight and is designed to be stacked without crushing the solid (agar or gelatin-based) medium in the lower dishes in the stack. The dishes can be made of low cost materials, preferably plastic, are designed to be readily molded, are sterilizable, and preferably can be disposed after use. Because of the incorporation of a biocatalytic means of removing oxygen, an enlarged headspace is possible. This enlarged headspace relieves the moisture condensation problems encountered with the Brewer Lid.
Moreover, the dish top of the culture dish in certain embodiments of the present invention, has a small dome or cavity designed to contain an anaerobic gas (such as CO2) generating pad or indicator strips to show the anaerobic state within the headspace of the closed culture dish. A variation of this dish design provides for additional removal of moisture from the dish as needed by placing pores in the bottom of the dish base. This feature prevents the build-up of excess condensate inside the dish which leads to flooding of the agar media surface. The pores are too small to let molten agar media flow out of the dish, yet they provide an exit for moisture. An oxygen intruding into the dish through these pores must pass through the media containing the oxygen reducing agent. This intruding oxygen is removed before it can diffuse to the top layer of media or into the headspace where it would interfere with growth of anaerobic microorganisms.
The culture dish, i.e., xe2x80x9cOxyDish(trademark)xe2x80x9d of the present invention, is designed for automated preparation of agar or gelatin-based media plates necessary for commercial production. When in the upright position, the dish can be readily filled with molten medium (such as a molten agar or gelatin-based media) without the sealing ring contacting the medium surface. When stored or used, the dish is placed into an inverted position. In this position, a seal (i.e. a media-ring seal) is formed by the contact of the sealing ring of the dish top with the media surface contained in the dish bottom when the media surface comes to rest on the sealing ring. This creates a headspace defined by the media surface, the inside wall of the sealing ring, and the inside top of the dish lid.
Furthermore, when the culture dish is utilized with the oxygen reducing agent such as a biocatalytic oxygen reducing agent, the oxygen reducing agent in the media reacts with the oxygen trapped in that headspace. This reaction renders the headspace sufficiently low in oxygen such that microorganisms affected by the presence of oxygen can grow on the media surface typically within 24 to 48 hours when the dish is incubated at 35xc2x0 C. to 37xc2x0 C. in an aerobic incubator. Any oxygen that intrudes into the dish around the media ring-seal or through the plastic is removed by the action of the reducing agent. The catalytic reducing agent facilitates the design and function of this dish.
The media suitable for use in the present invention includes any solid type media which can be inverted to form a media ring-seal. Solid media generally consists of liquid media which have been solidified (xe2x80x9cgelledxe2x80x9d) with an agent such as agar or gelatin. Examples of other known suitable gelling agents include alginate, gellan gum (xe2x80x9cGelrite(trademark)xe2x80x9d) and silica gel (xe2x80x9cPluronic Polyol F127(trademark)xe2x80x9d). The solid type media is of such a composition to support growth of anaerobes, microaerophiles and facultative aerobes.
Further, the culture dish, i.e., xe2x80x9cOxyDish(trademark)xe2x80x9d, of the present invention, is designed in certain embodiments so that it can be stacked in a stable configuration. The dish top has a stacking ring that interlocks with the adjacent dish top below it. The dish bottom, when the assembled dish is inverted and placed in a sealed position, rests (i.e., nests) between the two adjacent dish tops. The functionality of the dish to establish and maintain an anaerobic environment is preserved and protected in the stack. The stackability of the culture dish increases the efficient use of incubator space. Stackability is also important for the mechanized filling of these dishes and shipment of dishes or of finished pre-made, plates to the microbiologist or end user.
The culture dish of the present invention, simplifies handling anaerobes by the microbiologist or laboratory technician. The culture dish, i.e., xe2x80x9cOxyDish(trademark)xe2x80x9d, can be opened and closed several times while continuing to generate an anaerobic environment in the closed position. The specially designed culture dish significantly increases the microbiologist""s efficiency by reducing and simplifying the number of manipulations required to work with anaerobes. Furthermore, the microbiologist can now treat each culture dish and its microbial contents individually. This allows the microbiologist to make decisions based on his observations of each isolate or treatment, rather than having to wait for the slowest growing isolate in a group of culture dishes present in a sealable jar, bag, etc. In addition, the self-contained, environmentally controlled culture dish provides a secure environment for the microbe inside.
The foregoing has outlined some of the most pertinent objects of the invention. These objects should be construed to be merely illustrative of some of the more prominent features and applications of the intended invention. Many other beneficial results can be attained by applying the disclosed invention in a different manner or by modifying the invention within the scope of the disclosure. Accordingly, other objects and a more detailed understanding of the invention may be had by referring to the drawings, the detailed description of the invention and the claims which follow below.